1. Field of the Invention
This invention relates to measuring and testing of environmental and biological phenomena. More particularly, the present invention relates to methods and apparatuses for environmental monitoring and bioprospecting.
2. Description of Prior Art
Bioremediation is an effective, yet inexpensive biotechnology for removing organic and inorganic pollutants from contaminated environments. When targeting dissolved metals and radionuclides, the goal is to convert water-soluble, toxic species to insoluble, less toxic products. For example, uranium may be removed from contaminated groundwater and immobilized in the subsurface via the injection of carbon sources that stimulate the microbially induced precipitation of dissolved U(VI) in the form of insoluble U(IV). In this case, the contaminant is being treated “in place” and the process is being referred to as in situ bioremediation.
When designing in situ bioremediation strategies, it is essential to gain an understanding of the type, activity, and nutritional requirements of subsurface microbial communities present at a specific cleanup site. Microbial community information also is important for convincing regulatory agencies and stakeholders that the contaminant is being removed (or, in the case of metals, successfully immobilized in the subsurface) rather than being diluted or dispersed in groundwater.
Currently, the assessment of bioremediation potential at a given site is both labor- and cost-intensive. A typical approach for implementing bioremediation includes the following two steps: (1) Microcosm screening studies conducted in the laboratory to determine the extent of intrinsic bioremediation and to identify the type, quantity and frequency of carbon source injection that may be needed in order to accelerate the in situ bioremediation process; these experiments also serve to estimate contaminant removal rates but do not accurately reflect actual in situ removal rates due to the biases introduced by laboratory “bottle effects,” and (2) Microbial community profiles are obtained from microcosm and field samples to determine the microorganisms responsible for the desired biotransformation reactions; since most microorganisms fail to grow on laboratory media, culture-independent profiling techniques are commonly used, (e.g., 16S rDNA-based analyses).
Groundwater is the usual preferred sample matrix for profiling of microbial communities, as it is both readily available and inexpensive. Unfortunately, the lifestyle of a given target organism has a significant impact on one's ability to detect it in this matrix. In the extreme, a target organism pursuing a sessile lifestyle throughout its existence will be impossible to detect in groundwater at a site even if it is present at extremely high densities. Thus, groundwater monitoring alone may not accurately reflect the microbial community composition and dynamics of subsurface environments. Recently, solid-phase samplers were rediscovered as useful tools for overcoming some of these limitations.
In their simplest configuration, solid-phase samplers are nothing more than a physical surface incubated in an environment of interest for a period of time sufficiently long to allow for the colonization by microorganisms. Buried or submerged glass slides have been used extensively to collect microorganisms from soils, bioreactors and other environments. Following retrieval of such samplers, microorganisms are extracted and identified via the detection of biomarkers including DNA, phospholipids, fatty acids and respiratory quinones. An argument can be made that microorganisms collected with a solid-phase sampler are more representative of the metabolically active microbial community than those obtained by groundwater sampling because the sampling device requires the active physical attachment by the microorganisms to be captured. However, dead microorganisms, cell debris and DNA also may become entrapped. Highly sensitive tools (e.g., the polymerase chain reaction, PCR) can detect biomarkers in non-living material as well as those of metabolically active microbial community members.
Recently, stable-isotope markers have been used to distinguish metabolically active microorganisms from those being dormant or non-viable. Stable isotope probing (SIP) exploits the fact that the DNA of an organism growing on carbon-13 enriched carbon sources becomes 13C-labeled (“heavier”), thereby enabling one to resolve its DNA from the total community DNA by density gradient centrifugation. While representing a powerful research tool, stable isotope probing appears to have limited potential for being applied for routine biological monitoring since the technique is very time- and labor-intensive.
An alternative approach for the identification of microorganisms is to look for gene expression products (i.e., proteins) rather than for their characteristic DNA sequences. This can be done with the latest generation of mass-spectrometry instrumentation that offers sufficient speed and sensitivity, while also allowing for complete automation of the analysis process.
Matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), with its ability to induce desorption of protein biomarkers from intact bacteria, fungi, spores and viruses, provides a powerful and rapidly emerging technology for fast, portable and robust microorganism identification. MALDI-TOF-MS techniques are very rapid (<5 minutes analysis time per sample), have low sample volume requirements (<1 mL) and have a generic capability to identify microorganisms.
Robotic devices recently have been integrated with MALDI-TOF instruments to provide for automation of this analysis technique. The latest generation of commercially available robotics allows for the fully automated sample preparation and analysis, including preparation and imaging of 2D gels, harvesting and digestion of the protein spots, and application of the digests to multi-sample MALDI-TOF targets for analysis.
Despite all the prior art in this field, there still exists an ongoing need for improved testing methods and apparatus. For example, a methodology and technology that could integrate the above technologies so as to provide for both microbial community profiles and microcosm screening studies in a one-step, lower cost process would contribute greatly to this field. Ideally, such a fully developed methodology and technology would yield information on what types of organisms are present, which are alive and metabolically active, what type of nutrients and nutrient dosages should be used to accelerate bioremediation, and what in situ bioremediation rates would result.
Such a new methodology and technology should also prove to be quite valuable in other in situ applications; for example, in bioprospecting in saturated media, i.e., for the discovery of novel microorganisms, biochemical reactions, and natural products. Since less than an estimated one percent of environmental microorganisms are thought to be capable of growing and functioning under laboratory conditions, a new methodology and tool for exploring in situ microbial processes could effectively open the research door to a large fraction of the uncharted microbial world.
The underlying rationale of such an invention would be—since the majority of microorganisms do not tolerate the transfer from their natural habitat to the laboratory—to deliver the laboratory to the microorganisms. The impact of such an invention would be to benefit both human and environmental health by accelerating the discovery of novel microorganisms, enzymes and metabolic processes.
The present inventor has been working in this technical field and towards the development of such an improved methodology for some time. Much of his earlier research is applicable to the methodologies described herein. Most of this work has been documented in the scientific literature. See for example: Franklin, M. P., V. Madrid, S. Gregory, and R. U. Halden, “Spatial Analysis of a Microbial Community Mediating Intrinsic Reductive Dechlorination of TCE to cis-DCE at a DOE Superfund Site,” presented at the 103rd ASM General Meeting, Washington, D.C., May 18-22, 2003; Halden, R. U., B. G. Halden, and D. F. Dwyer, “Removal of dibenzofuran, dibenzo-p-dioxin, and 2-chlorodibenzo-p-dioxin from soils inoculated with Sphingomonas sp. strain RW1.” Appl. Environ. Microbiol., 65:2246-2249 (1999); Halden, R. U., E. G. Peters, B. G. Halden, and D. F. Dwyer, “Transformation of mono- and dichlorinated phenoxybenzoates by phenoxybenzoate-dioxygenase in Pseudomonas pseudoalcaligenes POB310 and a modified diarylether-metabolizing bacterium,” Biotechnol. Bioeng. 69:107-112 (2000); Halden, R. U., S. M. Tepp, B. G. Halden, and D. F. Dwyer, “Degradation of 3-phenoxybenzoic acid in soil by Pseudomonas pseudoalcaligenes POB310(pPOB),” Appl. Environ. Microbiol. 65:3354-3359 (1999); Colquhoun, D., E. S. Wisniewski, D., A. Kalmykov, and R. U. Halden, “Identification of Sphingomonas wittichii RW1 Through the Dioxin Dioxygenase Enzyme Using Mass Spectrometry. 104th General Meeting of the American Society for Microbiology, New Orleans, La., May 23-27 (2004); Halden, R. U., R. N. Cole, C. Bradford, D. Chen, and K. J. Schwab, “Rapid Detection of Norwalk Virus-like Particles using MALDI-TOF MS and ESI-MS/MS,” 51 st Meeting of the American Society for Mass Spectrometry, Montreal, Quebec, Canada, Jun. 8-12, 2003, http://www.inmerge.com/aspfolder/ASMSSchedule2.asp; Lowe, M., E. L. Madsen, K. Schindler, C. Smith, S. Emrich, F. Robb, and R. U. Halden, “Geochemistry and microbial diversity of a trichloroethene-contaminated Superfund site undergoing intrinsic in situ reductive dechlorination,” FEMS Microbiology Ecology 40:123-134 (2002); Vancheeswaran, S., R. U. Halden, K. J. Williamson, J. D. Ingle, and L. Semprini, “Abiotic and biological transformation of tetraalkoxysilanes and trichloroethene/cis-1,2-dichloroethene cometabolism driven by tetrabutoxysilane-degrading microorganisms,” Environ. Sci. Technol. 33:1077-1085 (1999); and Vancheeswaran, S., S. H. Yu, P. Daley, R. U. Halden, K. J. Williamson, J. D. Ingle, and L. Semprini, “Intrinsic remediation of trichloroethene driven by tetraalkoxysilanes as co-contaminants: results from microcosm and field studies,” Remediation 13/14:7-25 (2003). The teachings and disclosure of these works are hereby incorporated herein by reference.
3. Objects and Advantages
There has been summarized above, rather broadly, the background that is related to the present invention in order that the context of the present invention may be better understood and appreciated. In this regard, it is instructive to also consider the objects and advantages of the present invention.
It is an object of the present invention to provide an improved, lower cost method for environmental monitoring and bioprospecting.
It is another object of the present invention to provide an improved bioremediation assessment method and tool that will more effectively support the environmental restoration and long-term stewardship of contaminated sites. It is yet another object of the present invention to provide an improved bioremediation assessment method and tool that will enable the automated, large-volume, high-throughput analysis of bioremediation sites.
It is a further object of the present invention to provide a monitoring method, tool and analysis strategy that allow for the automated, rapid and simultaneous determination of the following parameters: (1) fluid quality and toxicity, (2) intrinsic bioremediation potential, (3) accelerated bioremediation potential following nutrient amendment, (4) effective bioaugmentation strategies for environmental cleanup, (5) turnover rates of natural compounds and environmental pollutants under natural and enhanced conditions, (6) in situ DNA synthesis and protein expression, (7) in situ growth/death rates and metabolic activity of native and introduced biological agents under natural and altered environmental conditions, (8) structure and dynamics of microbial communities indigenous to natural environments, and (9) identity and activity of microorganisms of potential value for use in biotechnology.
It is an object of the present invention to provide a monitoring method and tool that may be applied to assess the potential risk resulting from the release of nonindigenous microorganisms, pathogens and genetically engineered microorganisms into natural environments.
It is another object of the present invention to provide a method and tool that have potential value for discovering microorganisms, enzymes and natural products of relevance for the pharmaceutical industry and the biotechnology sector.
These and other objects and advantages of the present invention will become readily apparent as the invention is better understood by reference to the accompanying summary, drawings and the detailed description that follows.